Human kinesins and methods of producing and purifying human kinesins

Abstract

Described herein are methods of producing kinesins. In a preferred embodiment, the kinesins are produced from a prokaryote, most preferably, a bacterial cell. Bacterial expression offers several advantages over systems previously utilized, such as, for example, Bacculovirus. The yield of protein is higher, the cost of the expression setup is lower, and creation of alternative expression vectors is easier. The concern of copurifying a contaminating activity from the expression host is also eliminated since bacteria, in contrast to the bacculovirus expression system, do not have kinesin like proteins. Also described herein are purified kinesins, preferably unglycosylated and methods of use.

We claim:  
     
       1. A substantially pure unglycosylated human kinesin protein comprising a motor domain and at least three epitope tags. 
     
     
       2. The protein of  claim 1 , wherein said kinesin protein is selected from The group consisting of KSP or SEQ ID NO:29, CENP-E of SEQ ID NO:31, MKLPI of SEQ ID NO:27, HSET of SEQ ID NO:37, Kin2 of SEQ ID NO:25, Kif1A of SEQ ID NO:39 and MCAK of SEQ ID NO:33. 
     
     
       3. The protein of  claim 2 , wherein said kinesin protein is KSP of SEQ ID NO:29. 
     
     
       4. The protein of  claim 3 , wherein said kinesin protein consists of residues 1-491 of KSP of SEQ ID NO:29. 
     
     
       5. The protein of  claim 3 , wherein said kinesin protein consists of residues 1-360 of SEQ ID NO:29. 
     
     
       6. The protein of  claim 2 , wherein said kinesin protein in CENP-E of SEQ ID NO:31. 
     
     
       7. The protein of  claim 6 , wherein said kinesin protein consists of residues 2-340 of CENP-E of SEQ ID NO:31. 
     
     
       8. The protein of  claim 6 , wherein said kinesin protein consists of residues 2-405 of CENP-E of SEQ ID NO:31. 
     
     
       9. The protein of  claim 6 , wherein said kinesin protein consists of residues 2-465 of CENP-E of SEQ ID NO:31. 
     
     
       10. The protein of  claim 6 , wherein said kinesin protein consists of residues 2-488 of CENP-E of SEQ ID NO:31. 
     
     
       11. The protein of  claim 6 , wherein said kinesin protein consists of residues 2-329 of CENP-E of SEQ ID NO:31. 
     
     
       12. The protein of  claim 2 , wherein said kinesin protein is MKLPI of SEQ ID NO:27. 
     
     
       13. The protein of  claim 12 , wherein said kinesin protein consists of residues 4-433 of MKLPI of SEQ ID NO:27. 
     
     
       14. The protein of  claim 12 , wherein said kinesin protein consists of residues 4-494 of MKLPI of SEQ ID NO:27. 
     
     
       15. The protein of  claim 2 , wherein said kinesin protein is HSET of SEQ ID NO:37. 
     
     
       16. The protein of  claim 15 , wherein said kinesin protein consists of residues 152-519 of HSET of SEQ ID NO:37. 
     
     
       17. The protein of  claim 15 , wherein said kinesin protein consists of residues 151-519 of HSET of SEQ ID NO:37. 
     
     
       18. The protein of  claim 2 , wherein said kinesin protein is Kin2 of SEQ ID NO:25. 
     
     
       19. The protein of  claim 18 , wherein said kinesin protein consists of residues 166-532 of Kin2 of SEQ ID NO:25. 
     
     
       20. The protein of  claim 18 , wherein said kinesin protein consists of residues 195-532 of Kin2 of SEQ ID NO:25. 
     
     
       21. The protein of  claim 2 , wherein said kinesin protein is MCAK of SEQ ID NO:33. 
     
     
       22. The protein of  claim 21 , wherein said kinesin protein consists of residues 2574-617 to MCAK of SEQ ID NO:33. 
     
     
       23. The protein of  claim 21 , wherein said kinesin protein consists of residues 257-660 of MCAK of SEQ ID NO:33. 
     
     
       24. The protein of  claim 2 , wherein said kinesin protein is Kif1a of SEQ ID NO:39.